GITR induces up-regulation of p21 and PUMA in MM cells.

A) Total protein and RNA was extracted in 0, 3, 6, 12, 24 and 48 hours after treatment with GITRL (10ng/mL) from GITR- and GITR+ MM1.S cells. Both Protein and mRNA level of p53 have been evaluated by western blot and qRT-PCR. Actin was considered as the protein loading control. CT value of qRT-PCR was normalization to 18s. Mean ±SD. B) GITR- and GITR+ cells were exposed to GITR-L (0-5-10ng/mL) for 24 hrs. whole cellular protein was extracted and subjected to Western blot using anti-p21, -PUMA and -actin antibodies. C) GITR- and GITR+ MM1.S cells were exposed to GITR-L (5-10 ng/mL) for 24 hours respectively. Cell cycle has been evaluated by using PI staining and flow cytometry analysis. D) GITR- and GITR+ MM1.S cells were exposed to GITR-L (5-10ng/mL) for 24 hours. Whole protein lysates have been subjected to Western blot using anti-caspase-3, 9 and –tubuilin antibodies. E) GITR- and GITR+ MM1.S cells were exposed to GITR-L (5-10ng/mL) for 24 hours. Cells were stained by PI/Annexin V to evaluate apoptosis effect using flow cytometry. Mean ±SD.